Colloidal gold solutions (gold sols) are useful in immunochemistry, bioresearch, and medical diagnosis and in the physical and chemical sciences. A gold sol is a suspension of gold particles measuring about 5 to 150 nm in either a flake or particle shape. The shape and size of the particle is important in permitting the particle to remain in suspension. In other words, the particles need a high surface-to-volume ratio. This geometry allows the particle to be subject to the forces of Brownian movement rather than the force of gravity thereby allowing it to remain in suspension.
Gold sols are used in research and medicine as, primarily, a labeling agent. Gold sol can be bound to proteinaceous molecules and other compounds facilitating their use as a labeling agent. Because of the electron dense nature of the gold particles, gold sol-conjugated labels are reagents of choice in electron microscopy, light microscopy, flow cytometry, blotting, hybridization assays and rapid diagnostic tests.
Current manufacturing methods for gold sol and gold sol-based conjugates are time consuming and labor intensive and/or have poor quality and quantity yields. The methods are dependent on precise chemical and physical parameters and often require “clean-up” steps involving ultracentrifugation, filtering or other mechanical means to remove gold sol aggregates (e.g., see, U.S. Pat. No. 6,833,275 B1 to Nichtl, which is herein incorporated by reference; Geoghegan, W. D. and G. A. Ackerman, J. Histochem. Cytochem. 25, 1187-1200, 1977). It is believed that aggregation occurs because current manufacturing procedures result in supersaturation of the gold particles in the solution. Because of the supersaturation, the gold sol particles form “nuclei.” These nuclei facilitate aggregation in a process called nucleation. Controlling aggregation via optimized prior art manufacturing conditions is theoretically possible but in practice is hard to achieve (Chauduri, B. and S. Raychauduri, Manufacturing High-Quality Gold Sol, IVD Technology, March 2001). If not removed, the aggregates can cause the clumping of proteins (or other substances chosen to be gold sol-labeled) upon conjugation. These clumps then interfere with the protocol for which the gold sol conjugates are used.
Current manufacturing protocols also achieve relatively low yields. A concentration yielding an OD 530 nm reading of about 2 units is typical (see, e.g., U.S. Pat. No. 4,313,734 to Leuvering, which is herein incorporated by reference). Because of the labor intensity of the current manufacturing processes as well as the poor yields that are obtained, gold sol production is costly.
Additionally, gold sol conjugation is also not without problems. For example, conjugation of gold sol to thiol-containing molecules such as proteins often require the “coating” of the gold sol with n-alkanethiol and n-alkanethiol derivatives prior to conjugation (e.g., see, U.S. Pat. No. 5,294,369 to Shigekawa, which is herein incorporated by reference). These additional steps add even greater time and cost to an already expensive and labor-intensive undertaking.
What is needed are methods for the more efficient and economical production of gold sol and gold-sol thiol conjugates.